Devices and formulations

ABSTRACT

Methods are described of delivering biological material, which methods include the steps of providing a liquid formulation containing the biological material, supplying the liquid formulation to an outlet and subjecting liquid issuing from the outlet to an electrical field thereby causing electrohydrodynamic processing of the liquid without denaturing the biological material. In one example, the liquid formulation is provided by removing salts from a formulation containing biological material that does not denature in alcohol and then adding an alcohol to the formulation before supplying the liquid formulation to the outlet. An acid may be added to the liquid formulation before supplying the liquid formulation to the outlet.

[0001] This invention relates to methods and devices for processingliquids using electric fields, in particular but not exclusively tomethods and devices for comminuting relatively highly conductive liquidformulations such as aqueous formulations using electric fields.

[0002] A method of processing liquids using electric fields is describedin, for example, GB-A-1569707. In this method, which is known as theelectrohydrodynamic method (sometimes also referred to herein as “EHD”),liquid issuing from an outlet is subjected to an electric field suchthat the net electrical charge in the liquid as the liquid emerges intofree space or air counteracts the surface tension forces of the liquidand the repulsive forces generated by the like electrical charges resultin a cone and jet. Depending upon the liquid formulation, the liquid jetmay then, as described in GB-A-1569707, breakup into liquid droplets, ormay, as described in, for example, the applicant's WO 98/03267 (thewhole contents of which are incorporated by reference), break up to formsolid or gel-like particles or may form a continuous fibre which maybreak -up into short lengths (“fibrils”). The products resulting fromthe electrodynamic method are, for convenience, collectively referred toherein as “electrosols”.

[0003] This electrohydrodynamic method is particularly good atcontrolling the dimensions of the resultant product and provides anextremely efficient way of delivering drugs or medicaments to therespiratory system, for example to the pulmonary system, and to otherepithelial or topical surfaces such as wound surfaces as described in WO98/03267. Furthermore, as described in WO 98/03267 electrohydrodynamicmethods may be used to spray complex colloids, provided the colloid isinitially in a substantially liquid form.

[0004] The electrohydrodynamic method enables sprays or clouds ofdroplets (“aerosols”) to be produced in which the droplets aremonodispersed, that is they have a very uniform size and does not,unlike some conventional aerosol producing methods, require a propellantgas. This makes inhalers using the electrohydrodynamic method such asdescribed in the applicant's U.S. Pat. No. 4,962,885, U.S. Pat. No.6,105,877, U.S. Pat. No. 6,105,571, U.S. Pat. No. 5,813,614, U.S. Pat.No. 5,915,377 and WO 99/07478 (which enable delivery of at leastpartially electrically discharged droplets) and WO 00/35524 (whichenables delivery of electrically charged droplets) particularlyadvantageous because the absence of a gas propellant makes the inhalereasy to use as inhalation does not have to be timed with the expulsionof gas from the inhaler and the monodispersed nature of the aerosolcombined with the ability provided by the electrohydrodynamic method tocontrol the size of the droplets enables drugs or other medicaments tobe targeted to a particular region of the respiratory system, forexample a specific region of the lung. The whole contents of U.S. Pat.No. 4,962,885, U.S. Pat. No. 6,105,877, U.S. Pat. No. 6,105,571, U.S.Pat. No. 5,813,614, U.S. Pat. No. 5,915,377, WO 99/07478, and WO00/35524 are hereby incorporated by reference.

[0005] As more is understood about the way biological species operate,veterinary and medical treatments increasingly incorporate biologicalmolecules or material such as DNA, RNA, proteins, peptides, hormones,lipids, cytokines, etc. into therapies, treatments and prophylacticmedicaments such as vaccines. As used herein the term “biologicalmaterial” includes biological molecules, biological molecule fragmentssuch as DNA fragments and recombinant biomolecules, including proteinssuch as enzymes and other biological material of a similar size. Thesebiological materials vary in their complexity but some, particularlyproteins and DNA, are extremely sensitive to their immediatesurroundings and can easily be broken down or denatured which can reducetheir activity and even eliminate it altogether. The delivery ofbiological material also requires the occasional use of isotonic orbuffered liquid vehicles, and because such materials are often expensiveto produce, delivery systems must be as efficient as possible.

[0006] Traditional methods of atomising liquids of this kind, such asair-jet or ultrasonic nebulization, impart large shear forces on thecarrier liquid and hence also on the biological material inside. Shearforces of this magnitude are known to denature sensitive biologicalmaterials such as DNA or proteins and thus there is no readily availabledelivery method that is immediately suitable for therapies that use suchbiological materials.

[0007] The carrier liquids for the biological material mentioned aboveare generally aqueous and relatively highly conductive. Unfortunately,EHD is well known to have difficulty in spraying conductive liquids.Numerous patents and published papers indicate that the resistivity ofthe liquid to be sprayed must be above 10,000 Ohm.m. Liquids below thiswill spray, but there seems to be a cut-off at around 100 Ohm.m, belowwhich no aqueous-based formulation will spray in air. This is partly dueto the surface tension of water which is high, approximately 72 mN/m(milli Newtons per metre), and partly due to the polar nature of water,which makes any impurities such as a water soluble drug contributesignificantly to the liquid conductivity. This has meant thatnon-aqueous solvents such as ethanol tend to be used for EHD.

[0008] Furthermore, EHD comminution uses high voltages (1 KV and above)to break up liquid formulations by direct counteraction of the surfacetension of the liquid. The use of such high voltages raises severalpotential practical problems, namely: 1) that the electric field mightdirectly influence, denature or break up delicate, for examplebiological, materials in the liquid; 2) that breaking up the bulk liquidinto small droplets might physically denature such delicate biologicalmaterials through excessive shear forces; and 3) that air which breaksdown around the nozzle might create ozone which will react with anywater in the formulation to produce hydrogen peroxide which is itself astrong oxidant and the presence of which could lead to moleculardenaturation.

[0009] These problems have meant that EHD has to date only really beenpractical for small, robust molecules, such as salbutamol andbudesonide, which have good solubility in alternative solvents likeethanol. However, ethanol is not a good solvent for biological materialsbecause it can cause precipitation (as it does for DNA) and denaturation(as it does for delicate proteins).

[0010] In one aspect, the present invention provides a method ofenabling EHD to be used with highly conductive, generally aqueous,liquids.

[0011] In one aspect, the present invention provides a method ofenabling EHD to be used to dispense biological materials such as DNA,RNA, proteins, peptides, hormones, lipids, cytokines and recombinantbiomolecules.

[0012] In one aspect, the present invention provides a method ofcontrolling processing of liquid using EHD wherein the partial vapourpressure of a volatile component of a liquid is controlled in the regionadjacent to where the liquid is subject to the electric field.

[0013] In one aspect, the present invention provides a method ofcontrolling electrohydrodynamic comminution of an aqueous liquidformulation to produce droplets, wherein the air in the region intowhich the liquid issues is dried or dehumidified to cause evaporation toreduce the size of the droplets. This should facilitate production ofdroplets for supply to the respiratory system of babies and even verysmall mammals, such as mice.

[0014] In one aspect, the present invention provides an EHD inhalationapparatus capable of delivering biological material and other delicatemolecules or compounds such as drugs to humans and animals such as mice,

[0015] In one aspect, the present invention provides devices and methodswhich enable generation of respirable droplets containing delicate,aqueous-based molecules that is superior to any other method currentlyavailable.

[0016] In one aspect the present invention provides anelectrohydrodynamic comminution device which causes no denaturation ofbiological material or sensitive molecules, and thus benefits fromproducing comminutes with controlled diameter and charge.

[0017] In one aspect the present invention provides a method forspraying delicate molecules or biological material without denaturation.

[0018] In one aspect the present invention provides a method ofmodifying the product of EHD processing by air preparation that enablesproduct consistency (that is whether the product is solid, gel orliquid) and droplet size to be changed by affecting local evaporationcharacteristics for the liquid.

[0019] In one aspect the present invention provides a formulation forpreparing DNA in solution which adds ethanol without precipitating outthe DNA.

[0020] In one aspect the present invention provides a method of sprayingDNA by EHD wherein an acid (e.g. acetic acid) is added in order to makeDNA containing solutions sprayable at higher concentrations.

[0021] In one aspect the present invention provides a method of sprayingDNA by EHD wherein EDTA or Catalase is added to the liquid to be sprayedto prevent DNA degradation.

[0022] In one aspect the present invention provides a method of sprayingDNA by EHD wherein aqueous or high surface tension formulations are madesprayable by adding increased quantities of surfactants, generally at aconcentration significantly above the Critical Micelle Concentration.

[0023] In one aspect the present invention provides a method thatenables aqueous or highly conductive formulations to be sprayed by EHDby the addition of at least one long chain polymer (e.g. PVP, PVA, ethylcellulose) to the liquid to be sprayed.

[0024] In one aspect the present invention provides a method of enablingspraying by EHD of formulations which normally form unstable sprays byadding polymer (either a single polymer or a combination of polymers) tothe liquid to be sprayed.

[0025] In one aspect the present invention provides a method ofenhancing sprayability of a formulation by EHD by adding surfactant andpolymer to the liquid formulation to be sprayed.

[0026] In one aspect the present invention provides a method of sprayingbiological material by EHD wherein polymer is added to the biologicalmaterial-containing liquid formulation to be sprayed. This protects andstabilises the delicate biological material within the liquidformulation.

[0027] In one aspect the present invention provides a method of makingformulations sprayable by EHD at either polarity by the addition ofpolymers to the formulation.

[0028] This enables use of the twin nozzle or outlets devices describedin U.S. Pat. No. 6,105,877 and U.S. Pat. No. 5,915,377 to spray aqueousformulations.

[0029] In an embodiment, devices and methods are provided that enableelectrohydrodynamic comminution of aqueous liquids containing drumsand/or biological material to produce sprays or dispensions which areideal for respiration and delivery to the internal epithelia, includingthe lung, trachea, throat, mouth and nasal passages, and could also beadvantageous for all topical applications.

[0030] Embodiments of the present invention will now be described, byway of example, with reference to the accompanying drawings, in which:

[0031]FIG. 1 shows a schematic part-cross-sectional view of a firstexample of a dispensing device;

[0032]FIG. 2 shows a schematic part-cross-sectional view of a secondexample of a dispensing device;

[0033]FIGS. 3 and 4 show examples of use of a dispensing device as anoral and nasal inhaler, respectively;

[0034]FIG. 5 shows the droplet spectrum obtained using a methodembodying the present invention;

[0035]FIG. 6 shows a photograph of an electrophoretic gel plot of DNAtraces for comparing the amount of degradation obtained when a methodembodying the present invention is used to produce a nebulized oratomised droplet spray containing the DNA with the amount of degradationobtained when other methods are used to produce a nebulized or atomiseddroplet spray containing DNA;

[0036]FIG. 7 shows a photograph of an electrophoretic gel plot of DNAtraces, illustrating the lack of degradation for various DNA plasmidswhen a method embodying the present invention is used to produce anebulized or atomised droplet spray containing the DNA plasmid;

[0037]FIGS. 8 and 9 show graphs illustrating the effect on hydrogenperoxide degradation of the addition of EDTA and Catalase, respectively;

[0038]FIG. 10 shows a photograph of an example of comminuted matterproduced using a method embodying the invention;

[0039]FIG. 11 shows a photograph of another example of comminuted matterproduced using a method embodying the invention;

[0040] FIGS. 12 to 14 show block diagrams of different inhalationapparatus for delivering electrosol for inhalation;

[0041]FIG. 15 shows a perspective view of an EHD comminution chamber andinhalation chamber suitable for use in the inhalation apparatus shown inFIG. 12;

[0042]FIGS. 16 and 17 illustrate schematically and in cross-sectiondifferent examples of air adaptation units suitable for use in aninhalation apparatus;

[0043]FIG. 18 shows a plot illustrating the change in humidityconcentration in the inhalation chamber of the inhalation apparatusshown in FIG. 12 over time with and without the air adaptation unit;

[0044]FIGS. 19 and 20 illustrate schematically two versions of coldtraps that may be used as air adaptation units in an inhalationapparatus;

[0045]FIG. 21 shows a schematic part-cross-sectional view of a modifiedversion of the dispensing device shown in FIG. 1;

[0046]FIG. 22 shows a schematic part-cross-sectional view of a modifiedversion of the dispensing device shown in FIG. 2; and

[0047]FIG. 23 shows a graph illustrating the binding of rSP-D to Eagan4A LPS after incubation with various solvents.

[0048] Referring now to FIG. 1, there is shown a dispensing device 1suitable for use in methods embodying the invention to be described indetail below. The device 1 has a housing 2 which is generally formed ofan electrically insulative material. The housing 2 has an outlet 3 andis divided into first and second chambers 4 and 5. The second chamber 5has air inlets 2 a. The first chamber contains a voltage source 6 suchas a battery, a high voltage generator 7 for generating a high voltage(of the order of kilovolts) from the battery voltage and a reservoir 8for containing the liquid to be subjected to electrohydrodynamicprocessing. The reservoir is coupled to a, in this example, generallyinsulative liquid supply tube 9 which extends into the second chamber 5and has an outlet 10 in the second chamber 5. Liquid may be suppliedfrom the reservoir 8 to the liquid supply tube 9 under gravity or bymeans of a pump as described in any of U.S. Pat. No. 4,962,885, U.S.Pat. No. 6,105,877, U.S. Pat. No. 6,105,571, U.S. Pat. No. 5,813,614,U.S. Pat. No. 5,915,377, WO 99/07478 and WO 00/35524. A user operableswitch SW is provided to enable a user to couple the voltage source 6 tothe high voltage generator 7.

[0049] In this example a comminution site is provided by establishing ahigh voltage between a first conductive electrode 11 supported withinthe tube 9 and a second electrode 12 provided on the outer surface ofthe tube 9. The first and second electrodes are coupled to a highvoltage and an earth (ground) supply lead 13 and 14, respectively, ofthe high voltage generator 7.

[0050] When a user operates the switch SW to couple the voltage source 6to the high voltage generator 7 a high voltage (generally of the orderof kilo volts) is provided between the first and second electrodescausing an electric field to be established in a comminution region 20adjacent the outlet 10. Liquid issuing from the outlet is thus subjectedto this electric field such that the net electrical charge in the liquidas the liquid emerges into free space or air counteracts the surfacetension forces of the liquid and the repulsive forces generated by thelike electrical charges result in a cone and jet which, depending uponthe liquid formulation, may then break up into liquid droplets.

[0051] The dispensing device 1 produces an electrically chargedcomminution. Further details and modifications of this device can befound in WO00/35524.

[0052] Any of the comminution site arrangements described in any of U.S.Pat. No. 4,962,885, U.S. Pat. No. 6,105,877, U.S. Pat. No. 6,105,571,U.S. Pat. No. 5,813,614, U.S. Pat. No. 5,915,377, WO99/07478 and WO00/35524 may be used in place of the comminution site described above.Also, the device shown in FIG. 1 may be modified to enable at leastpartial discharge of the electrically charged comminution in any of theways described in U.S. Pat. No. 4,962,885, U.S. Pat. No. 6,105,571, U.S.Pat. No. 5,813,614, and GB-A-1569707.

[0053]FIG. 2 shows a dispensing device 1 a based on that described inWO99/07478 in which the second electrode 12 is replaced by a dischargeelectrode 12 a spaced from the tube 9 and in which a further ordeflection electrode 13 is provided. In this case the high voltagegenerator 7 is arranged to maintain the further electrode 13 at avoltage intermediate the voltages applied to the first and dischargeelectrodes 11 and 12 a. The further electrode 13 serves, as describedWO99/07478, to deflect the comminution away from the discharge electrode12 a until sufficient space charge has been generated by production of acomminution. The device 1 a may be modified to have the sameconstruction as any of the embodiments described in WO 99/07478 or anyof the modifications of the embodiments described in WO 99/07478.

[0054] The dispensing devices 1 and 1 a shown in FIGS. 1 and 2 may beused to dispense comminuted matter into a chamber or, especially wherethe comminuted matter is electrically charged, onto a surface. In thesecases the second electrode 12 shown in FIG. 1 may be omitted and anearth may be provided at or by the surface to which the spray isdirected. Also, the outlet 3 may as illustrated diagrammatically in FIG.3 be coupled to a mouthpiece 30 to enable oral inhalation of thecomminuted matter by a human user or may be adapted to be receivedagainst or slightly within a nostril of a human user as shown in FIG. 4to enable nasal inhalation.

[0055] Methods embodying the invention of enabling electrohydrodynamicprocessing (otherwise referred to herein as spraying) of liquids willnow be described.

[0056] Method 1

[0057] A method will now be described of enabling electrohydrodynamiccomminution of an aqueous solution containing biological material suchas DNA. EHD formulations often incorporate ethanol because it has a lowconductivity and a low surface tension. It can be added to mostformulations, but with DNA its addition conventionally causesprecipitation. In fact, the addition of ethanol is actually taught as ameans of precipitating DNA out of solution (see for example “MolecularCloning—Laboratory Manual—Vol3, 2^(nd) ed.” Sambrook, Fritsch, Maniatis;ppE10-15; Coldspring Harbour Press; 1989). We have however developed amethod that enables ethanol to be added to a DNA solution to make itsuitable for EHD.

[0058] DNA is usually stored with various buffer solutions which containthe salts of various chemicals. In this method, the vast majority ofthese salts are removed by dialysis to produce a relatively pure DNAsolution. After removal of these salts ethanol is added to produce aformulation which is 80% by volume of ethanol. Surprisingly this doesnot cause the DNA to precipitate out. The reservoir of a dispensingdevice such as that shown in FIG. 1 or 2 was then filled with theformulation and the dispensing device activated. The liquid was sprayedat a flow rate of 1 μl/s (micro litre per second) from, in this example,a liquid supply tube in the form of a single capillary tube nozzle. Thisformulation was found to. spray satisfactorily when the DNAconcentration was low (up to about 200 micro grammes per millilitre) butdid not spray satisfactorily at higher concentrations of DNA.

[0059] Further experiments were carried out with an ethanolconcentration of 70% and different ethanol concentrations up to 90%. Allwere found to spray satisfactorily with low DNA concentrations.

[0060] Method 2

[0061] Method 1 was repeated but with the modification that a smallamount (approximately 1 mM) of acetic acid was added to the formulationand sprayed at a flow rate of 0.5 ml/hr (millilitres per hour). Thisformulation was found to spray satisfactorily even when the DNAconcentration was significantly increased and satisfactory spraying wasachieved with DNA concentrations of 6 mg/ml. Similar results wereobtained with acetic acid concentrations from 0.2 mM up to 1 mM (milliMolar).

[0062] A practical rate of DNA nebulization was achieved without theprecipitation any conventional formulation with this concentration ofDNA would have caused. Also, the resulting EHD comminution had, as shownin FIG. 5, a very desirable droplet spectrum with a very narrow range ofdroplet diameters.

[0063] We have found acetic acid to be perfectly adequate for thispurpose, but other acids have been used such as nitric acid andhydrochloric acid. This formulation enables spraying of DNA but can alsobe used for any proteins that do not denature in the presence ofethanol.

[0064]FIG. 6 shows a electrophoretic gel plot for various DNA samplesafter spraying through different nebulizers. The outer two lanes showthe kilobase markers (Kb) that help quantify the results. The lanemarked pCIKβGal shows the plot for the DNA that has not been sprayed.The lanes marked ‘Jet’ and ‘Ultra’ correspond to the DNA collected afternebulization with a ‘Sidestream’ air-jet nebulizer (produced by MedicAid, of Bognor Regis, UK) and with a ‘Euroneb’ ultra-sonic nebulizer(produced by Medikare, of Germany). The smear of DNA below the originalposition (indicated by the pCIKβGal lane) in the Jet and Ultra lanesshows that the nebulization process resulted in significant damage tothe DNA. By contrast the lane marked ‘EHD’ which shows the DNA dispensedusing method 2 described above is practically identical to the originalDNA, indicating little or no damage by the nebulization process. Thisalso shows that the high voltages used during EHD comminution do notaffect the DNA.

[0065]FIG. 7 shows plots similar to FIG. 6 of results obtained usingmethod 2 for various different DNA plasmids. The results are grouped inpairs, where the left hand result for each pair represents the DNAbefore spraying whilst the right-hand plot shows the DNA after EHDcomminution using method 2 described above. The three plasmids shown arepCIKCAT which is 4.6 kilobases, pCIKCFTR.10 which is 9.2 kilobases, andpREP8βGal which is 14.2 kilobases. As can been seen dispensing the DNAusing method 2 above does not appear to affect any of these plasmids.

[0066] Methods 1 and 2 may be used to spray by EHD DNA, DNA fragmentsand other biological material that is not denatured by alcohols.

[0067] Method 3

[0068] In method 1 or 2, hydrogen peroxide is formed when ions createdduring the EHD comminution process react with the water in theformulation. Although the amount of hydrogen peroxide produced isextremely small, degradation caused by the peroxide can clearly be seenon the DNA. This degradation reaction is catalysed by the presence ofmetal ions in the formulation which are naturally present in minutequantities and are extremely difficult to get rid of. The amount ofdegradation is absolute and small and at DNA concentrations above 0.025mg/ml, we have found that the percentage degradation is hardlynoticeable. Consequently, for a therapeutic formulation, which mighthave a concentration 40 times greater than this or even more, theperoxide degradation can be neglected. However, where the concentrationof DNA being sprayed is low, for instance around 0.0025 mg/ml, theeffect on the DNA is significant.

[0069] In this method the formulation of method 1 or 2 was modified byadding either 50 mM ethylenediaminetetra-acetic acid (EDTA) or 40 nMCatalase.

[0070]FIGS. 8 and 9 show the percentage of supercoiled DNA (determinedby electrophoretic plot) in comminuted matter containing DNA collectedafter spraying using a formulation incorporating as an additive 50 mMethylenediaminetetra-acetic acid (EDTA) or 40 nM Catalase, respectively.Values are taken before and after spraying and for each formulationthere is very little immediate damage, that is very little change in theamount of supercoiled DNA. However, at a time about one hour afterspraying the results begin to diverge. Thus although the amount ofsupercoiled DNA in the collected comminuted matter produced using method3 (shown by the solid squares) remains virtually unchanged, the amountof supercoiled DNA remaining in the collected comminuted matter whichdoes not incorporate the additive (shown by the open or outline squares)begins to decrease. It is thus clear that the DNA sprayed without usingthe additive gradually deteriorates .

[0071] Although, as stated above, the amount of degradation isnegligibly small at high DNA concentrations so that method 3 is notnecessary at high DNA concentrations, where only low concentrations ofDNA are wanted the results show that DNA degradation can be completelyavoided by addition of a chelating agent such as EDTA or an enzyme suchas catalase.

[0072] Further experiments involving the spraying of proteins have alsobeen carried out and have proved that such molecules are also notdenatured during EHD comminution. For example, Trypsin, peroxidase andrecombinant lung surfactant-protein D have all been sprayed withoutdenaturation. Also other alcohols than ethanol, for example polyethyleneglycol 200 have been used.

[0073] Methods 1 to 3 may be used for any proteins or other biologicalmaterials that do not denature in ethanol or other alcohol egpolyethylene glycol 200.

[0074] Method 4

[0075] The introduction of ethanol or other co-solvents such aspolyethylene glycol 200 may in some circumstances cause denaturation andmay therefore be unacceptable.

[0076] This method provides another way of enabling comminution by EHDof aqueous formulations which may contain biological material. Until nowit has been practically impossible to use EHD to comminute aqueousformulations. This is due to the high surface tension and highconductivity of these formulations which tend to cause the air to breakdown in the vicinity of the comminution point or region, leading tolarge, sporadic, local electrical currents, which generally causecatastrophic failure of the EHD cone-jet.

[0077] Where possible surfactants can be added to the aqueousformulation solution to lower the surface tension. All surfactants havean intrinsic property known as the Critical Micelle Concentration (CMC)which is the concentration at which micelles form in the body of theliquid, and which conventionally corresponds to the minimumconcentration at which the surface activity of the surfactant ismaximised. However, experiments have shown that this concentration istoo little surfactant for EHD comminution, rather we have found that theconcentration must be increased to allow for a monolayer distributedover the high specific surface of the comminuted matter in addition tothe surfactant's CMC.

[0078] Experiments have been carried out using as the surfactantdidodecyldimethylammonium bromide whose molecular surface area isroughly 68 Å². The Critical Micelle Concentration (approximately 0.061%by mass) was insufficient to produce satisfactory comminution using thedispensing device shown in FIG. 1 or 2. However increasing thesurfactant concentration to a level sufficient to provide a monolayercoating enabled satisfactory comminution. Thus for typical droplets of1.5 μm in diameter at least approximately 0.4% ofdidodecyldimethylammonium bromide is required and a concentration ofaround 0.5% was effective to produce satisfactory EHD comminution orspraying of the aqueous formulation.

[0079] Also we have found that addition of 0.1% benzalkonium chloride inwater allows a liquid with a resistivity measuring only ˜60 Ohm.m tospray at 0.12 μl/s. Similarly, addition of 0.8% Tween 20 (apolyoxyethylene sorbitan derivative) allows a liquid with a higherresistivity of approximately 625 Ohm.m to spray at the same flow rate.

[0080] Other suitable surfactants have also been used such as Tween 80,Emulphogen (now known as Rhodosurf BC720 (polyoxyethylene 10 cetylether)) Brij 30 (polyoxyetbylene 4 lauryl ether).

[0081] Method 5

[0082] Unfortunately, despite these results there are still someproblems associated with using surfactants. These problems include:toxicity or irritation caused by the surfactants on sensitive epithelia;the fact the maximum flow rates are relatively low which leads toprotracted treatment times; the relative instability of these sprayssuch that the cone-jet can easily be disrupted by mechanical vibration;and the fact that surfactants, in practice, generally only enable EHDsprays of a positive polarity—at negative polarity air breaks down moreeasily and the reduction of surface tension is generally not sufficientto allow spraying by EHD.

[0083] In this method higher flow rates can be achieved with conductiveformulations containing biological material without the need forsurfactants. In this method a long-chain polymer that is soluble in theliquid to be sprayed is added to the formulation. Where that liquid iswater, suitable polymers include polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP),, polyhyaluronate, polysucrose, and otherpolysaccarides, such as starch, cellulose and chitin and chemicalderivatives thereof, poly amino acids, modified collagen and itsderivatives. Other polymers that are, or can be made to be soluble inthe liquid formulation (water soluble where the formulation is anaqueous formulation) may be used provided they are of a suitable lengthto effect the dynamic relaxation constant of the liquid. Alsocombinations of polymers or different weights of the same polymer may beused. For example PVP 40000 may be used in combination with PVP 360000in the spraying of aqueous formulations.

[0084] As an experiment a highly conductive aqueous formulation with aresistivity of around 5 Ohm m was selected as the liquid to be sprayed.The surface tension of the liquid was roughly 70 mN/m and withoutformulation modification this liquid would not spray in air using EHDcomminution. In this experiment, the aqueous formulation was found tospray satisfactorily at flow rates of up to around 1.5 ml/hour uponaddition of only 2% of PVP of molecular weight 360000.

[0085] The molecular weight of the selected polymers is importantbecause the benefits polymers such as these can provide will only beapparent if the molecular weight is suitably high. We have found thatdifferent polymers require different molecular weights and that thebeneficial effects change gradually as higher molecular weights areselected. As a general rule, the higher the molecular weight of a givenpolymer, the lower the concentration needed to optimally modify aformulation for spraying. As the concentration of the polymer isincreased, the product of comminution changes from being droplets tobeing fibres. The concentration at which this transition takes placeillustrates the variation in effect that can be expected from differentpolymer types. As an example of using different molecular weights of thesame polymer, formulations of ethanol containing PVP360000 in increasingconcentration were sprayed and compared with ethanol formulationscontaining PVP40000. The formulations containing PVP360000 began toproduce fibres at a concentration of 35 mg/ml, whereas the transitionfor the PVP40000 containing solutions was at 260 mg/ml. It follows,therefore, that less PVP360000 than PVP40000 would be needed tostabilise a given formulation. As an example of using differentpolymers, a formulation containing 70% Ethanol and 30% salty water(water containing NaCl at 0.5M) was sprayed with PVP 360000 andPVA125000 respectively. With PVP360000, the transition concentration ofpolymer was 55 mg/ml. With PVA125000, the transition from droplets tofibres occurred at a concentration of 30 mg/ml. Similarly, it has beenshown that combinations of different molecular weight polymers followthe pattern. For example, a formulation containing 70% Ethanol and 30%salty water (water containing NaCl at 0.5M) was sprayed containing bothPVP40000 and PVP360000 (in 50:50 ratio), the transition concentration ofcombined polymer in this case was 70 mg/ml.

[0086] In another experiment a formulation was used which consisted of70% ethanol, and 30% water. The water also contained some salt (NaCl) at0.5M to mimic the effect of adding an active molecule, biologicalmaterial or drug, lowering the resistivity to 4.7 Ohm.m. 0.2 g/10 ml PVPof molecular weight 360000 was added to the formulation which wassprayed using a device such as that shown in FIG. 1 or 2. FIG. 10 showsa close up photograph taken from a microscope slide on which theresultant comminuted material was collected. The droplets formed are ofa respirable size, measuring roughly 5 μm or below. The verticaldimension of the photograph is 75 μm.

[0087] In another experiment 10 mg/ml of PVA of molecular weight 125000was added to a formulation containing 70% ethanol and 30% water byvolume, where the water contained 0.5M salt(NaCl) and the formulationhad a resistivity of 5.5 Ohm m. FIG. 11 shows a picture similar to FIG.10 taken from a microscope slide on which the resultant comminutedmaterial was collected. Again the vertical dimension of the photographis 75 μm and the droplets produced were respirable.

[0088] Similar results were obtained with PVA of molecular weight 125000in concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 grammes per 10millilitres of liquid formulation and with PVP of molecular weight360000 in concentrations of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 grammes per10 millilitres of liquid formulation consisting of 70% ethanol and 30%0.5 Mol water-NaCl solution to mimic the presence of an activeingredient in the form of biological material. Similar results have alsobeen obtained by adding a polymer combination of 50% PVP of molecularweight 40000 and 50% PVP of molecular weight 360000 in concentrations inthe range 0.5 g/10ml to 0.8g/10 ml of formulation. Concentrations ofpolymers above a certain level will result in fibre formation ratherthan droplets formation with the actual level being dependent on thepolymer and the formulation. Typically this level is about 0.5 g/10 mlfor PVP 360000, 0.6 g/10 ml for a 50:50 mix of PVP of molecular weights40000 and 360000 and 0.25 g/10 ml for PVA of molecular weight 125000.

[0089] Incorporation of a long chain polymer enables in a dynamicreduction of the formulation's relaxation constant to be achievedallowing higher flow rates with conductive liquids. Furthermore, andmore importantly, the polymer-incorporating liquids will spray at eitherpolarity.

[0090] Method 6

[0091] In this method, a combination of polymer and surfactant is usedto optimise the EHD spray and electrosol product.

[0092] A formulation consisting of a solution of salt (NaCl) dissolvedin water at 0.5M was used as an example. This will not spray using EHDcomminution. However, after adding 1.7% (17 mg/ml) PVA with a molecularweight of 125000 and 1% (10 mg/ml) Tween 20, the formulation (whoseproperties were: resistivity=4.3 Wm; surface tension=32 mN/m; andviscosity=13 cP) sprayed at flow rates between 0.2 and 0.6 ml/hour.

[0093] Similar results may be obtained with any of the polymersmentioned above.

[0094] Method 7

[0095] Our experiments have shown that delicate molecules such asproteins can be stabilized by the addition of polymers.

[0096] In this method, various proteins were conjugated withpolyethylene glycol (PEG) prior to spraying. This was found to increasetheir stability, to protect the proteins from mechanical stresses and toenable them to tolerate more variation in their liquid carrier orvehicle. Other polymers having properties similar to PEG produce similarresults.

[0097] Examples of experiments carried out to spray trypsin, peroxidaseand insulin will now be described.

[0098] Clinically available proteins such as insulin and growth hormoneare currently delivered by injection, and cannot be administered by theoral route, since they are digested and inactivated in thegastrointestinal tract and a significant portion are metabolised in theliver. It is therefore important to develop a pulmonary delivery systemto enable a wide range of these proteins to be delivered to the lungs.Insulin was used to determine the effects of spraying proteins with EHDtechnology.

[0099] Enzymes are essential in all cells, and belong to a large anddiverse group of proteins. They function as biological catalysts invirtually all biochemical reactions in biological systems. Trypsin is aproteolytic digestive enzyme that found in pancreatic juice of mammals.Peroxidase is another type of enzyme that presents in plants andanimals, especially mammalian spleen and lung, which acts on hydrogenperoxide and organic peroxides. These two enzymes were therefore used,as model systems, to determine the effects of spraying proteins with EHDtechnology.

[0100] Method 8

[0101] Examples of formulations using PEG200 will now be described.

[0102] Trypsin

[0103] Trypsin was first dissolved in sodium phosphate (NaPO₄) bufferand then formulated with PEG200 to make the final formulation: trypsinin 20% sodium phosphate 80% PEG200. 100 μl of this formulation wassprayed into a 35 mm polystyrene tissue culture dish containing 2 mlNaPO₄ buffer, with a flow rate of 1.5 μl/sec at +12.1 kV. A platinumloop immersed in the NaPO₄ buffer was used as the earth. After sprayingthe trypsin/NaPO₄ solution was removed by pipette from the culture dishfor enzyme assaying immediately after spraying, and 1, 2, 3, 4 and 5days later. The whole experiment was repeated four times and resultsshowed that 99.53% of the trypsin activity was retained after spraying.Therefore, it is concluded that the EHD spraying technique (withoutdischarging in this example) does not result in loss of enzyme activitywhen PEG200 is incorporated in the trypsin formulation.

[0104] Peroxidase

[0105] Peroxidase was formulated in 20% peroxidase diluent (containingpotassium phosphate buffer, bovine serum albumin and triton X-100) and80% PEG200. As with trypsin, 100 μl of this formulation was sprayed intoa tissue culture dish containing 2 ml peroxidase diluent, with a flowrate of 1 μl/sec at +11.9 kV. Again, a platinum loop immersed in theperoxidase diluent was used as the earth. Based on a singleexperiment, >90% of the peroxidase activity was retained four days afterspraying with the EHD technique (without discharging).

[0106] PEG (polyethylene glycol) can thus be used to stabilise proteinsto facilitate EHD.

[0107] Method 9

[0108] A method of enabling EHD spraying of formulations containingalcohol and biological material will be described.

[0109] Addition of PEG200 has, as set out above been shown to increasethe stability of proteins; however, in terms of toxicity, 80% PEG200 inthe formulation is a high level for inhalation, therefore alternativesolvents were considered. To this end, trypsin and peroxidase wereformulated with propylene glycol (often used in conjunction with wateror glycerol). Also, to determine if ethanol could be a potential solventfor spraying enzymes, formulations containing ethanol were also tested.

[0110] Trypsin was formulated in:

[0111] a) 20% NaPO₄ buffer, 80% propylene glycol [sprayable with flowrates of 0.6-2.4 μl/sec at voltages of +(8.1-8.6) kV],

[0112] b) 20% NaPO₄ buffer, 40% propylene glycol, 40% ethanol [sprayablewith flow rates of 0.6-1 μl/sec at +8.32 kV],

[0113] c) 20% NaPO₄ buffer, 60% propylene glycol, 20% ethanol [sprayablewith flow rates of 0.6-1 μl/sec at voltages of +(7.42-7.62) kV,].

[0114] Solutions (b) and (c) turned milky immediately after they weremade.

[0115] Peroxidase was formulated in:

[0116] d) 20% peroxidase diluent 80% propylene glycol [sprayable withflow rates of 0.6 μl/sec at +9.5 kV],

[0117] e) 20% peroxidase diluent, 40% propylene glycol, 40% ethanol,

[0118] f) 20% peroxidase diluent, 60% propylene glycol, 20% ethanol.

[0119] Solutions (e) and (f) were only sprayable with flow rates of0.01-0.02 μl/sec; corona resulted when the flow rate was higher. Thesolutions turned milky immediately after they were made.

[0120] Proteins are usually dissolved in buffer solutions that containsalts of various chemicals. Solutions turned milky when they wereformulated with alcohol, suggesting that precipitation of salts by thealcohol might have occurred. Results from a preliminary experimentshowed that phosphate buffer solution indeed turned milky when alcoholwas added. Also, a trypsin assay (see Appendix) was performed to testwhether the enzyme was stable after mixing with alcohol. The enzymeactivity was determined at 0, 1.5, 3, 4.5 and 6 hours after formulation.Results showed that at each time point, similar enzyme activity wasdetected in formulations containing 20% sodium phosphate buffer and 80%PEG 2000, and 20% sodium phosphate buffer and 80% ethanol, when comparedwith the formulations containing sodium phosphate buffer only. Theseresults show that the activity was retained in formulations containingalcohol.

[0121] Method 10

[0122] A method of spraying insulin by EHD will now be described.

[0123] Porcine insulin was formulated with three different formulations:

[0124] 7. 0.5% Emulphogen, 5% sodium phosphate (NaPO₄) buffer, 30%glycerol and 64.5% water

[0125] 8. 0.2% 0.01 M HCl, 19.8% sodium phosphate buffer and 80% PEG200

[0126] 9. 20% 0.01 M HCl and 80% PEG200

[0127] 100 μl of each formulation was sprayed into a 35-mm polystyrenetissue culture dish containing 2 ml NaPO₄ buffer. A platinum loopimmersed in the NaPO₄ buffer was used as the earth. After spraying, theinsulin/NaPO₄ solution was removed by pipette from the culture dish forinsulin testing with the ELISA method.

[0128] Results: Formulation Flow rate Voltage 0.5% Emulphogen 0.22μl/sec +6.6 kV 5% sodium phosphate buffer 30% glycerol 64.5% water 0.2%0.01 M HCl* 1 μl/sec +9.54 kV 19.8% sodium phosphate buffer 80% PEG20020% 0.01 M HCl 1 μl/sec +9.24 kV 80% PEG200

[0129] It was thus found that insulin can be formulated in threedifferent sprayable formulations. The activity of insulin after sprayingwas determined by the ELISA method.

[0130] Trypsin, peroxidase and insulin can thus be delivered using EHDin various formulations.

[0131] Other Methods

[0132] Experiments have been also carried out to spray surfactantproteins using EHD. Pulmonary surfactant plays an important role inlowering the surface tension at the alveolar epithelial lining, bypreventing the collapse of alveoli during gas exchange. Failure ordeficiency of these proteins is associated with respiratory diseasessuch as respiratory distress syndrome (RDS). RDS remains the most commoncause of neonatal mortality. The administration of exogenous surfactantto newborn infants who have RDS is becoming an established therapeuticapproach.

[0133] Allergic reactions triggered by pollen grain/dust mites, and lunginfections caused by respiratory syncytial virus (RSV) are significantproblems worldwide. So far, no safe and efficient drugs are availablefor the prevention or treatment of these diseases. There is strongevidence that the lung surfactants (SPs), such as SP-A and SP-D, areinvolved in innate immunity, in which they bind to and remove allergenssuch as pollen, house dust mite droppings and pathogens such as virusesand fungi.

[0134] There are two main types of exogenous surfactant proteinscurrently available—natural and synthetic. Natural surfactants areobtained from animals or humans by means of alveolar wash or fromamniotic fluid. They have the advantage of having all the necessaryingredients for effective function to regulate surface tension; however,their collection is not time and cost effective. Also, they carry a riskof contamination with infectious viral agents. On clinical grounds,natural surfactants have greater efficacy than synthetic ones, perhapsdue to the protein content of natural surfactants. Therefore,recombinant lung surfactant proteins were developed and these sensitivemolecules can be delivered to the lungs for prevention and treatment ofrespiratory diseases, such as bronchial asthma, bronchiolitis andpneumonia, and enhancement of the immune defence in lung transplants.

[0135] The current method used to deliver proteins to the lungs of, forexample, mice is an invasive technique—intra tracheal instillation. Inthis method, hypodermic needles are inserted into the trachea of mice,through which proteins are delivered to the lungs. The insertion ofhypodermic needles to the trachea can easily damage the surroundingtissues, which might elicit unnecessary immune responses in the mice.Also, protein solutions delivered by the intra tracheal instillation arenot administered as droplets, but probably in aggregated form.Therefore, proteins are unlikely to reach the lower respiratory tract.

[0136] Another way to deliver proteins is by using liquid droppers,whereby proteins are delivered intra-nasally to animals such as mice.Similar to the intra tracheal instillation, one significant drawback ofthis delivery method is that it cannot deliver proteins accurately andtherefore results in an inefficient transferring of proteins.

[0137] In contrast to intra tracheal instillation, EHD enables materialto be delivered gently to the lungs of mice and humans, without damagingthe tissue along the respiratory tract. EHD also enables accuratedelivery of material because the size of the electrosol can bewell-controlled and so set to optimise the deposition zone in the lungsin dependence on the mass and shape of the droplets. For example EHD maybe used, as can be seen from FIG. 5, to produce a monodispersed dropletspray in which the droplets all have substantially the same diameterwith that diameter having a value in the range 1.0 to 10 microns forhumans and proportionally smaller for smaller animals. In addition EHDenables the resultant droplets to be electrically charged, electricallydischarged or partially electrically discharged as described in at leastsome of the patents and published applications mentioned above andretention of a small electrical charge may be advantageous, particularlyfor deposition in the terminal airways and alveoli.

[0138] Examples of EHD spraying of surfactant proteins

[0139] Recombinant SP-D (rSP-D) protein was formulated in threeformulations (a), (i) and (j) for spraying by EHD using the device asdisclosed in WO 99/07478. The further electrode was at earth (ground)potential. The formulation details and maximum flow rates obtainablewere: First electrode Flow rate, Discharge electrode Voltage Formulation(ml/h) voltage (kV) (kV) (a) 3% PVA (100 k in mol 1.7 2.36 −2.89 wt), 1%Tween 20, 96% 0.5 M NaCl (i) 2% PVA (100 k in mol 7.25 2.95 −3.26 wt),1% Tween 20, 48.5% 0.5 M NaCl, 48.5% glycerol (j) 2% PVA (100 k in mol 23.03 −3.08 wt), 1% Tween 20, 48.5% 0.5 M NaCl, 48.5% PEG200

[0140] The three formulations (a), (i) and (j) were tested for theireffect on the SP-D molecule without spraying. Recombinant SP-D was addedto each of the samples and assayed to test activity in each formulation,run against a control sample, Eagan 4A. A graph showing the activitiesof each mixture is shown as FIG. 23. The activity of the rSP-D in theformulations (a), and (j) was only slightly reduced. However, the SP-Din the formulation (i) did not produce a positive signal. It was notproven whether the glycerol actually denatured the rSP-D or simplyinterfered with the assay mechanism.

[0141] rSP-D in Aqueous Formulation

[0142] Based on the success with aqueous PVA and surfactant formulationsdescribed above, 0.1 mg/ml of rSP-D protein was formulated in 3% PVA(100 k in mol wt), 0.1% Tween 20 and 96.9% PBS. This formulation wassprayed at a flow rate of 1 ml/hr. The spraying was performed as asimple point-to-plane (nozzle tip to collection surface) experiment,using a device similar to that shown in FIG. 1 but without the secondelectrode and using as the liquid supply tube or nozzle a Delrin tipwith no central vane. The spray was collected in a petri dish using anearthed platinum wire loop. The point-to-plane distance was 20 mm. Deadspace was minimized, using 1 ml syringes and IV tubing, to conserve thesample since two repeats were performed for each formulation. A bioassaywas performed to determine whether the proteins remain fully active, ordenatured by the shear forces of the EHD process. Results showed that noadverse effects on the rSP-D due to EHD spraying occurred.

[0143] EHD spraying of SP-A and SP-D in vitro has also been achievedwith a near mono-dispersed spray or cloud being formed and nodenaturing. Controlled droplet charge has also been achieved by the useof a twin nozzle EHD device as described in WO 94/12285, the wholecontents of which are hereby incorporated by reference.

[0144] The above described experiments have shown that delicatematerials such as biological material, for example DNA, proteins andenzymes, can be subjected to electrohydrodynamic comminution withoutcausing electric-field induced denaturation, because the liquid carriersor vehicles of the formulation to be sprayed are more conductive thanconventional EHD formulations (i.e. have resistivities less than 10,000Ohm.m or more preferably 1000 Ohm.m). This extra conductivity helps toreject the external electric fields and thus protects againstelectric-field induced denaturation. Also, the shear forces occurringduring the EHD process are exceedingly low. The current wisdom is thatthe EHD cone which is the electric-field induced transition regionbetween bulk liquid and comminuted electrosol is highly turbulent with atoroidal eddy current set up by the electric field (Hayati I, 1985—Ph.D. Thesis, Imperial College London). However, the amount of turbulenceis related to the conductivity and viscosity of the liquid as well asthe physical dimensions of the comminution region or point. Theconductive formulations described here make a significant reduction tothe shear forces exerted on the liquid, and these can be reduced furtherby increasing the viscosity or reducing the diameter of the base of theEHD cone. Overall, we calculate that the liquids used experience shearforces during the EHD process that are around 1000 times lower thanair-jet and ultra-sonic nebulizers for the same size droplets.

[0145] The above methods may, as set out above, be implemented with anEHD dispensing device such as that shown in FIG. 1 or 2. This EHD devicemay, as will be described below, be incorporated into an inhalationapparatus that enables control over environmental conditions.

[0146]FIG. 12 shows schematically one example of an inhalation apparatussuitable for facilitating inhalation of droplets by small laboratoryanimals such as mice.

[0147] A pump 101 drives air through the inhalation apparatus at aconstant rate. The air from the pump travels through an air adaptationunit 102 into an EHD comminution device or unit 103. The air andcomminuted matter then travel into an inhalation chamber 104 where thesmall animals, generally mice, are placed to breathe the mist ofdroplets. Exhaust air exits via a filter 105 into the atmosphere.

[0148] In this example, the air adaptation unit 102 is designed toremove vapour from the incoming air.

[0149]FIG. 16 shows a cross-sectional view of one possible airadaptation unit 102, which comprises a U-tube 110 filled with desiccatedsilica gel 111. Air flows in through an inlet port 120 of the U-tube, isforced to pass through the silica gel 111 held in the U-tube and exitsthough an exit port 113. Both inlet and exit ports are secured and madeair tight by means of flexible bungs 114 made from rubber or anothersuitable material. In this way moisture is removed from the incoming airbefore it enters the EHD comminution unit 103. This enhances dropletevaporation enabling the production of smaller droplets. It will beevident to a person skilled in the art that other materials can beplaced in the U-tube to remove gases and vapours to help reduce dropletsize.

[0150]FIGS. 19 and 20 show two alternative cold traps which are suitablefor use as the air adaptation unit 102 for the removal of vapour fromthe air. In FIG. 19 a metal rod 311 is inserted between the inner andouter chambers of the inhalation box 104. The rod is dipped into liquidnitrogen 321 or another very cold liquid or solid held in a beaker 331,so that the rod becomes very cold. The part of the rod protruding insidethe inhalation box will condense out any vapour, and will significantlyreduce the vapour concentration throughout the box by simple vapourdiffusion. FIG. 20 shows an alternative cold trap which runs in asimilar fashion to a household refrigerator. Here a suitable refrigerantsuch as Freon-12 (dichlorodifluoromethane) is pumped around a metal tube401 by a compressor 411. The Freon-12 is allowed to expand across avalve 431 which significantly reduces its temperature and cools themetal tube. This inhalation apparatus can be adjusted during theexperiment to automatically maintain a specific vapour concentration ifcoupled to a suitable monitoring device (not shown). Other suitable coldtraps will be evident to a person skilled in the art.

[0151]FIG. 15 shows an example of a suitable inhalation chamber 104 formice for use in the inhalation apparatus shown in FIG. 12. The chamberconsists of a box 20 is made from Perspex having a perforate innerchamber 201 held symmetrically inside a sealed outer chamber 202 (apartfrom an air exhaust to which the filter 105 is coupled). The EHDcomminution unit or device 103 is mounted in the centre of the box lidto direct comminuted material into the perforate inner chamber 201. Theinhalation chamber 104 may have ambient modification means for modifyingand controlling evaporation or absorption of droplets produced by theEHD comminution unit 103 to enhance evaporation from the droplets tofacilitate droplet size reduction. The ambient modification means may beprovided by placing absorbent material, such as silica gel or activecharcoal, in the gap between the inner and outer chambers 201 and 202 toabsorb any unwanted vapour from the inhalation chamber so as to promoteevaporation from the surface of droplets produced by the EHD comminutionunit to reduce their size.

[0152]FIG. 18 shows how the humidity of the inhalation chamber 104 canbe radically altered over time by addition of 150 g of silica gel in theinhalation chamber. In this case the box was being sprayed with a 20%water, 80% ethanol mix by volume at a rate of 2.4 μl/s. It is clear fromthe graph that the presence of the silica gel ensures the relativehumidity in the box does not rise much above 40% for the entire durationof the experiment. It should be noted, however, that this experiment wascarried out with no animals in the box. Their breathing generates asubstantial amount of water vapour, and so cold traps such as thoseshown in FIGS. 19 and 20 may be introduced in between the inner andouter chambers to remove water vapour and other vapours from the airinside the inhalation chamber 104.

[0153] Typical air flow rates for this inhalation apparatus can varyfrom 0 to 60 L/min and above, but are preferably as low as possible,whilst maintaining a suitable oxygen level in the inhalation chamber. Inan experiment, ten mice were placed in the inhalation chamber 104, thepump was arranged to provide a flow rate of 4 l/min (litres/minute) ofair and the EHED comminution unit 103 was arranged to spray aformulation as described above in Method 2 with a DNA concentration of 1mg/ml at a flow rate of 2.4 μl/s. 17.3 mg of DNA was comminuted by theEHD comminution unit 103 over 2 hours and roughly 6 μg was retained inthe lungs of the mice.

[0154] This inhalation apparatus offers away of controlling the dropletdiameter and charge accurately down to the sort of sizes suitable formice (e.g. 1-3 μm and below). This inhalation apparatus thereforeenables the production of respirable aerosols for mice and similar smalllaboratory animals which would otherwise be extremely difficult due totheir small anatomy and non-compliant breathing facilitating the in vivotesting of the toxicity and activity of a therapy on animals such asmice (which have very different requirements from humans for inhalationdue to their radically different anatomy) as the necessary precursor forany human pulmonary therapy.

[0155] The use of chambers for the forced respiration of aerosols iswell known. However, conventional inhalation apparatus are inefficientin terms of the mass of product actually inhaled by the animal comparedwith the amount delivered. For instance, an air jet nebulizer whichproduces droplets of 1 μm, a suitable diameter for inhalation by mice,must be run at an air throughput of around 6-8 L/min (litre/minute).Consequently, it is difficult to build up a very high concentration ofthe aerosol in the air since it is continually diluted by the aircreating the aerosol in the first place. By contrast, in the inhalationapparatus described above the EHD comminution unit 103 creates thedroplets and only a relatively small air flow (<1 L/min) is required tomaintain the oxygen concentration in the inhalation chamber 104. Thismeans the electrosol can be concentrated by an order of magnitude, andthus the inhalation apparatus is an order of magnitude more efficientthan with a conventional air jet nebulizer. Ultrasonic nebulizers canalso be used without air flow. However, they also denature sensitivebiological material; they cannot be used with viscous liquids, (such asconcentrated solutions of DNA); and they easily block when creatingdroplets of around 1 μm.

[0156]FIG. 13 shows schematically an inhalation apparatus designed foruse with larger animals or human beings. In this inhalation apparatusthe inhalation chamber 104 is replaced by an intermediate chamber 106plus either a mask 107 as shown, which can fit over the mouth and noseof a larger animal such as a human (for example as shown in FIG. 3) or amouthpiece or intra tracheal tube. The mask can be any standardinhalation mask such as is available from Medic Aid, Bognor Regis, UK,Item Reference: 1100 System 22 face mask. The intermediate chamber 106may be based on the inhalation chamber 104 shown in FIG. 15 and so maybe provided with the modification means for modifying and controllingevaporation or absorption of droplets produced by the EHD comminutionunit 103 prior to delivery to the mouthpiece or mask 107.

[0157]FIG. 14 shows schematically an example of an inhalation apparatusintended for use by human beings. In this case the EHD comminution unit,intermediate chamber and mask are replaced by a single EHD inhalationdevice 108, such as described in WO99/07478. It should be noted thatthis inhalation apparatus could function perfectly well without the pump101 or air adaptation unit 102, although the use of the air adaptationunit may be advantageous to enable droplet size reduction as describedabove.

[0158] As described above the air adaptation unit 102 and possibly alsothe inhalation or intermediate chamber 104 or 106 are arranged to removevapour from the ambient air to facilitate evaporation from dropletsurfaces to reduce the droplet size. There are, however, circumstanceswhere it may desirable to increase the vapour pressure of a solvent orvolatile component in the air in which or in the vicinity of which theEHD process takes place. For example the solvent vapour pressure may beincreased to hinder evaporation to, for example, slow down or inhibitsolidification of the liquid issuing from the outlet of the EHDcomminution unit 103. This is particularly useful where the liquidformulation contains polymer because it enables solidification to beprevented and so allows droplet formation for higher polymerconcentrations than would otherwise be possible.

[0159]FIG. 17 shows a cross-sectional view of a bubble chamber which maybe incorporated into the air adaptation unit 102 (and/or the inhalationor intermediate chamber) and can be used to increase the partial vapourpressure, for example saturate the air which enters the EHD comminutionunit 103, with vapour of any solvent used in the liquid formulation.Referring to FIG. 17, air is pumped in through the inlet port 115 andbubbles through the liquid 116 which is held in a container 117. The airthen exits through the exit port 118. Both ports are held in place byair tight bungs 119 made from rubber or other suitable material.Experiments have shown that an aqueous formulation containing a polymercan be prevented from drying out and forming a solid product by usingsuch a bubble chamber containing as the liquid 116 warm water.

[0160] Where, as shown in FIG. 15, the EHD comminution device 103 isinserted into an inhalation or intermediate chamber, then the airadaptation unit 102 may be arranged to control the humidity of the airin that chamber rather than or in addition to in the EHD comminutiondevice 103 so that a separate modification means is not necessary.

[0161] As described above the diameter of droplets formed by the EHDcomminution unit 103 may be controlled by controlling the ambient inwhich the droplets are formed (by use of the air adaptation unit and/orby controlling the ambient in the inhalation or intermediate chamber 104or 106 as described above). Thus the droplet size can be reduced byenhancing evaporation of water (or volatile component such as a solventin the formulation, for example ethanol) from the droplets by decreasingthe partial vapour pressure of the water (or volatile component) in theambient. Thus drying the ambient air to reduce its humidity as describedabove will increase evaporation of water from the droplets and so reducetheir size. Conversely increasing the humidity (using for example thebubble chamber shown in FIG. 17) or increasing the partial vapourpressure of a volatile component of the formulation would hinderevaporation and so hinder droplet size reduction. In addition, where theliquid formulation contains a substance that is hygroscopic, dropletscan also take on water during flight and increase in diameter.

[0162]FIGS. 21 and 22 show modified versions of the EHD devices shown inFIGS. 1 and 2, respectively. These devices 1 b and 1 c differ from thoseshown in FIGS. 1 and 2 in that a second reservoir 50 is provided coupledby a pipe 51 to a solvent container in the form of a shroud or collar 52surrounding the liquid outlet tube 9. The reservoir 50 contains a supplyof solvent which is supplied by the pipe 51 to the shroud as a ratewhich substantially matches evaporation of solvent from the container52. In this example the container 52 contains an absorbent material suchas felt. It will of course be appreciated that although the liquidsupply tube 9 provides a convenient place to mount the container 52, thecontainer 52 could be mounted at any convenient place within the secondchamber 5. These modified EHD devices may be used alone or as the EHDunit 103 in any of the inhalation apparatus described above to increasethe solvent vapour pressure in the second chamber and can replace thebubble chamber shown in FIG. 17, enabling the air adaptation unit to beomitted. Generally the solvent will be a solvent used in the liquidformulation being sprayed, such as ethanol, for example.

[0163] It may also be possible where removal of vapour is required touse the shroud or collar (without the reservoir 50) to contain anappropriate desiccant (for example silica gel to dry the air in thesecond chamber to facilitate solvent evaporation from droplets to reducetheir size, also enabling omission of the air adaptation unit wheredrying of the ambient air is required.

[0164] In the above described examples the EHD process results indroplets. Where the liquid formulation contains a polymer then theamount of polymer may be such that comminution does not occur but rathera continuous fibre is formed which may break up into fibrils. Generallysuch formulations would not be used for inhalation purposes (unless thefibre breaks up into small fibrils) but may be used for topicalapplication to wound surfaces or the interior of the mouth, for example.In these cases, the modified devices shown in FIGS. 21 and 22 may beused to control the rate at which the liquid issuing from the liquidsupply tube solidifies to control the process of fibre formation ratherthen to prevent solidification entirely.

[0165] Where the liquid formulation contains a polymer, particularlywhere it contains a relatively large amount of polymer, a classicalTaylor cone will generally not be produced, rather the emerging liquidwill form a rather more hemi-spherical or blob-like shape at the liquidsupply tube outlet. The term cone as used herein is intended to coverboth the classical Taylor cone and these shapes.

[0166] Methods, devices and inhalation apparatus embodying the inventionmay be used to deliver biological material, water-soluble drugs ormedicaments, vaccines and other delicate molecules to internal orexternal bodily surfaces including the lungs, trachea, throat, mouth,nasal passages, eyes and skin and wound surfaces.

1. A method of delivering biological material, which method comprisesproviding a liquid formulation containing the biological material,supplying the liquid formulation to an outlet and subjecting liquidissuing from the outlet to an electric field thereby causingelectrohydrodynamic processing of the liquid without denaturing thebiological material.
 2. A method of delivering biological material,which method comprises providing a liquid formulation containing thebiological material, supplying the liquid formulation to an outlet andsubjecting liquid issuing from the outlet to an electric fieldsufficient to cause comminution of the liquid to produce dropletscontaining the biological material without denaturing the biologicalmaterial.
 3. A method according to claim 1 or 2, which comprisesproviding the liquid formulation by removing salts from a formulationcontaining biological material that does not denature in alcohol andthen adding an alcohol to the formulation before supplying the liquidformulation to the outlet.
 4. A method according to claim 3, whichfurther comprises adding an acid to the liquid formulation beforesupplying the liquid formulation to the outlet.
 5. A method according toclaim 4, wherein the acid is acetic acid.
 6. A method according to claim3, 4 or 5, wherein the alcohol is ethanol or polyethylene glycol.
 7. Amethod according to claim 1 or 2, which comprises providing the liquidformulation by adding surfactant to a formulation containing thebiological material before supplying the liquid formulation to theoutlet.
 8. A method according to claim 1 or 2, which comprises providingthe liquid formulation by adding polymer to a formulation containing thebiological material before supplying the liquid formulation to theoutlet.
 9. A method according to claim 1 or 2, which comprises providingthe liquid formulation by adding surfactant and polymer to a formulationcontaining the biological material before supplying the liquidformulation to the outlet.
 10. A method of delivering biologicalmaterial by processing a relatively highly conductive liquid such as anaqueous formulation containing the biological material, which methodcomprises adding polymer to the liquid, then supplying thepolymer-containing liquid to an outlet and subjecting polymer containingliquid issuing from the outlet to an electric field sufficient to causeformation of a cone and jet.
 11. A method according to claim 8, 9 or 10,wherein the polymer has a molecular weight of at least
 40000. 12. Amethod according to any one of claims 8 to 11, wherein the polymer isselected from the group consisting of PVA, PVP, polyhyaluronate,polysucrose and other polysaccharides such as starch, cellulose andchitin and chemical derivatives thereof, Palaemon acids, modifiedcollagen and derivatives thereof.
 13. A method according to any one ofclaims 8 to 11, which comprises adding 0.2 g/10 ml of PVP of molecularweight 360000 as the polymer.
 14. A method according to claim 8, 9 or10, wherein the polymer added is PVP with a molecular weight of 360000at a concentration in the range 0.1-0.6 g/10 ml.
 15. A method accordingto claim 8, 9 or 10 wherein the polymer added is PVA with a molecularweight of 125000 at a concentration in the range 0.1 to 0.7 g/10 ml offormulation.
 16. A method according to claim 8, 9 or 10, wherein thepolymer added is PVA with a molecular weight of 125000 at aconcentration of 10 mg per ml.
 17. A method according to any one ofclaims 10 to 16, which also comprises adding surfactant to theformulation.
 18. A method according to claim 7, 9 or 17, which comprisesadding as the surfactant a material selected from the group consistingof didodecyldimethylammonium bromide, benzalkonium chloride, Tween 20,Tween 80 and Brij
 30. 19. A method according to any one of claims 1 and3 to 18, wherein the electric field causes comminution of the liquid.20. A method according to any one of claims 8 to 18, wherein the amountof polymer added is such that subjecting polymer containing liquidissuing from the outlet to the electric field causes formation of afibre or fibrils.
 21. A method according to any one of claims 1 to 20,wherein the liquid contains biological material selected from the groupconsisting of DNA, DNA fragments, plasmids, RNA,, proteins, peptides,hormones, lipids, enzymes and cytokines.
 22. A method of enablingelectrohydrodyamic processing of an aqueous formulation containingbiological material, which method comprises removing salts from theaqueous formulation, then adding an alcohol and an acid to theformulation prior to subjecting liquid issuing from an outlet to anelectric field sufficient to cause formation of a cone and jet.
 23. Amethod according to claim 22, which comprises adding as the alcohol atleast one of ethanol and polyethylene glycol.
 24. A method according toclaim 22 or 23, which comprises adding as the acid acetic acid.
 25. Amethod according to claim 21, 22 or 23, wherein the alcohol adding stepcomprises adding alcohol to provide a formulation which is 70%, 80% orup to 90% by volume alcohol.
 26. A method according to any one of claims21 to 25, which comprises adding from 0.2 to 1 mM of acid in the acidadding step.
 27. A method according to any one of claims 22 to 26,wherein the biological material consists of at least one of DNA, DNAfragments, plasmids, RNA, proteins, peptides, hormones, lipids, enzymesand cytokines.
 28. A method of enabling electrohydrodyamic processing ofan aqueous formulation containing at least one of DNA, DNA fragments andplasmids, which method comprises removing salts from the aqueousformulation, then adding an ethanol and acetic acid to the formulationprior to subjecting liquid issuing from an outlet to an electric fieldsufficient to cause formation of a cone and jet.
 29. A method accordingto claim 28, which comprises adding ethanol to provide a formulationwhich is up to 90% by volume ethanol.
 30. A method according to claim28, which comprises adding ethanol to provide a formulation which isapproximately 70% or 80% by volume ethanol.
 31. A method according toclaim 28, 29 or 30, which comprises adding from 0.2 to 1 mM of aceticacid.
 32. A method of enabling electrohydrodyamic processing of arelatively highly conductive liquid such as an aqueous formulation,which method comprises adding surfactant to the liquid to provide asurfactant concentration significantly greater than the critical micelleconcentration in the liquid prior to subjecting liquid issuing from anoutlet to an electric field sufficient to cause formation of a cone andjet.
 33. A method of processing a relatively highly conductive liquidsuch as an aqueous formulation, which method comprises comprises addingsurfactant to the liquid to provide a surfactant concentrationsignificantly greater than the critical micelle concentration, thensupplying the surfactant-containing liquid to an outlet and subjectingsurfactant-containing liquid issuing from the outlet to an electricfield sufficient to cause formation of a cone and jet.
 34. A methodaccording to claim 32 or 33, which comprises adding as the surfactant amaterial selected from the group consisting of didecyldimethylammoniumbromide, benzalkonium chloride, Tween 20, Tween 80 and Brij
 30. 35. Amethod according to claim 32 or 33, which comprises adding as thesurfactant approximately 0.5% by mass of didodecyldimethylammoniumbromide.
 36. A method according to claim 32 or 33, which comprisesadding as the surfactant 0.1% by mass of benzalkonium chloride in water.37. A method according to claim 32 or 33, which comprises adding as thesurfactant 0.8% by mass of Tween 20 in water.
 38. A method according toany one of claims 32 to 37, wherein the liquid contains biologicalmaterial such as at least one of DNA, DNA fragments, plasmids, RNA,proteins, peptides, hormones, lipids, enzymes and cytokines.
 39. Amethod according to any one of claims 22 to 38, which comprises adding achelating agent or catalase to the formulation.
 40. A method accordingto any one of claims 22 to 39, wherein the electric field causescomminution of the liquid.
 41. A method of delivering biologicalmaterial by electrohydrodyamic processing of a liquid formulationcontaining biological material, which comprises conjugating thebiological material with PEG prior to electrohydrodyamic processing. 42.A method according to any one of claims 22 to 41, wherein the liquidcontains biological material selected from the group consisting of atleast one of DNA, DNA fragments, plasmids, RNA, proteins, peptides,hormones, lipids, enzymes and cytokines..
 43. A method according to anyone of the preceding claims, which comprises controlling the vapourpressure of a solvent of the liquid formulation in an environment intowhich the liquid issues from the outlet.
 44. A method of processingliquid which comprises supplying liquid to an outlet, subjecting liquidissuing from the outlet to an electric field to cause formation of atleast one of a fibre, fibrils or droplets and controlling the partialvapour pressure of a constituent of the liquid in the environment intowhich the liquid issues.
 45. A method of processing an aqueous liquidformulation, which comprises supplying the liquid to an outlet,subjecting liquid issuing from the outlet to an electric field to causecomminution of the liquid to produce comminuted matter and controllingthe humidity at least in the vicinity of the outlet to control the sizeof the comminuted matter.
 46. A method of processing an aqueous liquidformulation, which comprises supplying the liquid to an outlet so thatthe liquid issues into air, subjecting liquid issuing from the outlet toan electric field to cause comminution of the liquid to produce dropletsand drying the air in the environment into which the liquid issues or atleast in the vicinity of the outlet to cause evaporation of liquid toreduce the size of the droplets.
 47. A method of processing a liquidcomprising at least one polymer and at least one volatile component,which comprises supplying the liquid to an outlet, subjecting liquidissuing from the outlet to an electric field to cause formation of atleast one of a polymer fibre and fibrils and controlling the partialvapour pressure of the or at least one of the volatile constituents ofthe liquid in the environment into which the liquid issues or at leastvicinity of the outlet to control the formation of the fibre or fibrils.48. A method according to claim 43 or 47, which comprises supplying theat least one volatile component to the environment into which the liquidissues or at least to the vicinity of the outlet to control the partialvapour pressure of the at least one volatile component.
 49. A methodaccording to claim 48, which comprises providing around the outlet ashroud or collar for supplying the volatile component.
 50. A methodaccording to claim 49, which comprises supplying additional volatilecomponent to the shroud or collar.
 51. A method according to claim 49 or50, wherein the shroud or collar comprises an absorbent material.
 52. Amethod of electrohydrodyamic processing of a relatively highlyconductive liquid such as an aqueous formulation containing thebiological material, which method comprises adding polymer to the liquidprior to subjecting liquid issuing from an outlet to an electric fieldsufficient to cause formation of a cone and jet.
 53. A method ofprocessing a relatively highly conductive liquid such as an aqueousformulation containing the biological material, which method comprisesadding a polymer to the liquid, then supplying the polymer-containingliquid to an outlet and subjecting polymer containing liquid issuingfrom the outlet to an electric field sufficient to cause formation of acone and jet.
 54. A method according to any one of claims 44 to 53,wherein the liquid contains biological material selected from the groupconsisting of at least one of DNA, DNA fragments, plasmids, RNA,proteins, peptides, hormones, lipids, enzymes and cytokines.
 55. Amethod according to any one of claim 8, 9 or 10 to 16, which comprisessubjecting the liquid formulation to an electric field of negativepolarity.
 56. A method according to any one of claims 8, 9 or 10 to 16,which comprises supplying the liquid formulation to two liquid outletsand subjecting liquid issuing from the two different outlets to oppositepolarity electric fields.
 57. A method according to claim 1 or 2, whichcomprises conjugating the biological material with polyethylene glycol(PEG).
 58. A dispensing device for carrying out a method in accordancewith any one of claims 1 to 57, the device having: a liquid formulationreservoir; a liquid supply tube coupled to the reservoir and having aliquid outlet; and voltage applying means for subjecting liquid issuingfrom the liquid outlet to an electric field to cause liquid issuing fromthe outlet to form a cone and jet.
 59. A dispensing device, the devicehaving a housing containing: a liquid formulation reservoir; a liquidsupply tube coupled to the reservoir and having a liquid outlet; voltageapplying means for subjecting liquid issuing from the liquid outlet toan electric field to cause liquid issuing from the outlet to form a coneand jet; and supplying means for supplying solvent or volatile componentto the interior of the housing to increase the partial vapour pressurethereof.
 60. A device according to claim 59, wherein the supplying meanscomprises a collar or shroud surrounding the liquid supply tube.